Movements of matter and energy modify the spatial distributions of these quantities. The present invention uses the fact that such redistributions of energy and matter in space may induce local variations of the electromagnetic (EM) properties. Therefore devices able to probe electromagnetic properties can be used to monitor energy and matter fluxes.
Such type of interactions between EM radiations and matter or energy in movement are commonly used at a large and very large range in astronomy and environmental sciences. For example, the Doppler effect (or Doppler shift) can be used to measure speeds with Light Detection And Ranging (LIDAR) systems. At a smaller range, but still over the centimeter scale, flow meters exist based on optical methods that use Doppler velocimetry, or the Fresnel drag effect, or the property of light scintillation, but such devices requires relatively long probing distances, or even in some cases, the presence of scattering particles in the fluid. The present invention offers solutions to study such type of interactions at a the microscopic scale, thanks to the fact that apparatus according to the present invention measures preferably two type of electromagnetic properties that are highly sensitive to matter and energy densities, even over very short probing distances: the phase shifts of an electromagnetic radiation, and the refractive index.
The present invention will describe in more details an example of application that uses digital holographic microscopy (DHM) to measure ionic currents across the membrane of biological cells. For this particular application, the present invention is based on previous works, see e.g. EP1451646 (Marquet et al.), that have shown that DHM enables to obtain quantitative measurements of the phase shift induced by living cells. For cells, the phase shift can be regarded as a powerful endogenous contrast agent, which contains information about the cell morphology and about intracellular content related to the electromagnetic properties of the cell, in particular refractive index of the cell. The present invention use the fact that temporal variations of the cellular phase shift can be correlated with an electrical activity of cells and describe a method for evaluating associated trans-membrane ionic currents.
For its particular application in the field of cellular trans-membrane ionic currents monitoring, the present invention uses the fact that cellular stimulations inducing ions exchanges (currents) between the intra- and extra-cellular medium, are likely to affect the diffusion of water through the plasma membrane. Therefore changes in intracellular water concentration can be correlated with trans-membrane currents, and as water concentration and refractive index are directly linked, refractive index changes affecting cellular phase shift, can be correlated with ionic currents. Current techniques for online monitoring of this phenomenon (essential to maintain the ionic homeostasis) are scarce. For example, at a macroscopic scale, magnetic resonance imaging (RMI) studies have shown that the diffusion of water in the brain could monitor local increases in neural activity, see e.g. “Direct and fast detection of neuronal activation in the human brain with diffusion MRI” by D. Le Bihan et al, in Proceedings of the National Academy of Sciences of the United States of America 103, pages 8263-8268 in 2006. At the cellular level, imaging techniques based on the transmittance of light (intrinsic signal imaging) have demonstrated that physiological as well as pathological conditions could be associated with changes in cell-volume, see e.g “Optically recorded response of the superficial dorsal horn: dissociation from neuronal activity, sensitivity to formalin-evoked skin nociceptor activation” by J. Lee et al, in Journal of neurophysiology 94, pages 852-864, in 2005. However, all these imaging techniques provide mainly qualitative information on the water flow associated with cell activity. With the present invention the water flow through the cell membrane can be precisely measured with a high sensitivity and a high temporal resolution enabling real-time measurements. From such measurements, ionic currents can be derived very simply and very efficiently.
For its particular application in the field of ionic currents monitoring and measurements, the present invention offers several advantages compared to classical methods of electro-physiology. In particular, the present invention enables to analyze several cells simultaneously (multi-site recording) with a single detector (e.g. a video camera), while standard methods requires costly and complex instrumentations involving several electrodes. In addition, the present invention enables to measure currents in a non-invasive way without electrodes. Moreover, the present invention enable to determine unambiguously the direction of ionic fluxes, providing a clear differentiation between inward and outward currents, while electro-physiology fails in determining if an outward current is due to an outflow of cations (e.g. K+), or to an inflow of anions (e.g. Cl−), and inversely if an inward current is due to an inflow of cations, or to an outflow of anions.
Compared to optical electrophysiological techniques that use molecules capable of emitting light in response to their electrical environment, such as voltage sensitive dyes and fluoresceing proteins, the present invention offers the great advantage that bioelectric activity can be probed without use of contrast agents. Moreover, the present invention is directly sensitive to currents, while actual optical electrophysiological techniques are mainly voltage sensitive. In addition the present invention can be used to monitor optically electrical activities involving ions species for which no reliable optical dyes exist at present, such as chloride ions, which are of particular interest since they have important physiological roles, for instance in the central nervous system.